Liposomes Part B

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Introduction......Page 8
Substrates......Page 9
Asymmetric Incorporation of Glycosylphosphatidylinositol Lipids into Large Unilamellar Vesicle Outer Lipid Monolayers......Page 11
Enzyme Assays......Page 12
Vesicle Aggregation......Page 14
Intervesicular Lipid Mixing......Page 16
Intervesicular Mixing of Inner Monolayer Lipids......Page 17
Intervesicular Mixing of Aqueous Contents......Page 19
Phospholipase-Induced Vesicle Leakage......Page 21
Concluding Remarks......Page 24
Introduction......Page 25
Mechanism of Action of Secretory PLA2 at Membrane Interface......Page 26
Choice of Experimental System......Page 27
Charge......Page 28
Phospholipid Phase......Page 29
Calcium Concentration......Page 30
Summary......Page 31
Small Unilamellar Vesicles......Page 32
Snake Venom and Pancreatic......Page 34
pH-Stat......Page 35
Fluorescent Fatty Acid-Binding Proteins......Page 36
Thin-Layer Chromatography......Page 37
Assays of Binding......Page 38
Fluorescence Resonance Energy Transfer......Page 39
Probes of Membrane Phase and Order......Page 42
Pyrene-Labeled Probes for Assessing Changes in Lipid Distribution......Page 44
Fluorescence Imaging of Giant Unilamellar Vesicles......Page 46
Preparation of Giant Unilamellar Vesicles......Page 47
Binding Images......Page 49
Visualization of Hydrolysis......Page 50
Visualization of Changes in Membrane Properties......Page 52
Acknowledgments......Page 54
Introduction......Page 55
Preparation of Annexin V......Page 58
Phospholipid Vesicle Preparation......Page 59
Microelectrophoresis......Page 60
Fluorescence Measurements......Page 62
Protein Binding to Vesicles......Page 63
Principles of Fluorescence Correlation Spectroscopy......Page 65
FCS Instrumentation......Page 66
FCS Experiments......Page 67
Binding of Annexin V on PC, PS, and PA Liposomes at pH 7.4 and 4.0......Page 68
Discussion......Page 69
Introduction......Page 72
Strategies for Reconstitution of Membrane Proteins into Liposomes......Page 73
Dialysis......Page 75
Dilution......Page 76
Polystyrene Beads......Page 77
Mechanisms of Proteoliposome Formation and Efficiency of Reconstitution......Page 78
Preparation of Preformed Pure Liposomes......Page 80
Addition of Protein......Page 82
Parameters to Be Varied......Page 85
Functional Activity......Page 88
Protein Incorporation......Page 89
Protein Orientation......Page 90
Size Distribution......Page 91
Permeability......Page 92
Conclusion......Page 93
Introduction......Page 94
Isolation of Membranes Harboring Overexpressed SecYEG or YidC Protein......Page 96
Purification of SecYEG Complex......Page 97
Purification of YidC......Page 98
Blue Native Polyacrylamide Gel Electrophoresis......Page 99
Reconstitution of Membrane Proteins into Proteoliposomes......Page 100
Rapid Dilution......Page 101
Purification of SecA......Page 102
Production and Purification of proOmpA......Page 103
Translocation of Fluorescently Labeled proOmpA......Page 104
Acknowledgment......Page 106
General Aspects......Page 107
Subdivision according to Size......Page 108
Subdivision according to Structure......Page 109
Venom Components......Page 110
Mode of Action: Multiple Steps Leading to Pore Formation......Page 111
Liposomes as Convenient Model System......Page 113
Characterization of Liposome Systems......Page 114
Pore Formation Studies with Liposomes......Page 115
Detecting Toxin Binding......Page 116
Infrared and Ultraviolet Spectroscopy to Detect Changes in Secondary tructure and Folding......Page 117
Methods to Detect Formation of Oligomers......Page 119
Release of Fluorescent Molecules......Page 121
Pore Diameter Estimation and Passage of Neutral Molecules......Page 123
Measurements of Membrane Potential......Page 125
Demonstrating Formation of Toxin Oligomers......Page 127
Effects of Toxin on Lipid Organization......Page 129
Conclusions and Perspectives......Page 130
Acknowledgments......Page 132
Questions to Be Asked when Studying Antimicrobial Peptides with Liposomes......Page 133
Selectivity of Toxicity......Page 134
Lipid Binding......Page 135
Centrifugation......Page 136
Titration Calorimetry......Page 137
Structural Analysis......Page 138
Dye Leakage......Page 139
Lipid Flip-Flop......Page 140
Planar Bilayers......Page 141
Concluding Remarks......Page 142
Introduction......Page 143
Pure Fatty Acid Vesicles......Page 145
Mixed Fatty Acid-Alcohol Vesicles......Page 146
Mixed Fatty Acid-Polyol Vesicles......Page 147
Microscopy......Page 148
Procedure......Page 149
Methods of Encapsulation......Page 151
Removal of Nonentrapped Material......Page 152
Critical Issues during Separation......Page 153
Uptake of Externally Added Solutes......Page 154
Procedure......Page 155
Enzymatic Reactions Captured in Liposomes......Page 156
Polynucleotide Phosphorylase (PNPase)  /Qb Replicase......Page 158
Relevance to Early Forms of Cellular Life......Page 159
Acknowledgments......Page 161
Introduction......Page 162
Liposomes of Defined Lipid Composition......Page 165
Liposome Extrusion......Page 166
Buffers Used in Sedimentation, Fluorescence, and Light-Scattering Experiments......Page 167
Sedimentation Assay of Membrane Translocation of Arf1 and Rac on Liposomes......Page 168
General Requirements for Fluorescence, Fluorescence Resonance Energy Transfer, and Light-Scattering Experiments......Page 169
Activation and Deactivation Assays Based on Tryptophan Fluorescence......Page 170
FRET Assay to Monitor Membrane Translocationof Arf1 on Liposomes......Page 174
Light-Scattering Assay to Monitor Assembly of COPII Coat......Page 175
Acknowledgments......Page 177
Introduction......Page 178
Buffers and Salts......Page 179
Membrane Vesicles......Page 181
Membrane Potential: Long-Wavelength Oxonol VI Dye......Page 182
Diffusion Potential and Analysis of Filling/Emptying Kinetics......Page 186
Multilabeling Experiments......Page 188
Liposomes and Encapsulation of Nonpermeant Dyes......Page 189
Fluorescence Measurements......Page 190
Calibration of the pH Dye Pyranine......Page 191
Calibration of K+ Dye PBFI......Page 192
Ion Fluxes and Permeability Coefficient......Page 195
Acknowledgment......Page 198
Introduction......Page 199
Liposome Preparation......Page 201
Huge Unilamellar Vesicles: Injection Method......Page 202
Large Unilamellar Vesicles by Reversed-Phase Evaporation......Page 203
Interdigitation Fusion Vesicles......Page 204
Centrifugation Assay with Direct Determination of Protein......Page 205
Determination of Membrane-Bound Annexin by Light Scattering......Page 207
Direct Determination of Membrane-Bound Annexin by Fluorescence......Page 208
Indirect Determination of Membrane-Bound Annexin Using Phospholipase A2 Activity Assay......Page 210
Binding Kinetics Studied by FRET and Light Scattering......Page 211
Determination of Calcium Stoichiometry in Ternary Annexin-Calcium-Vesicle Complex......Page 212
Stability Assay......Page 213
Continuous Monitoring......Page 216
Annexin Effects on Liposome Aggregation as Monitored by Turbidity......Page 218
Monitoring of Fusion by Phospholipid Intermixing......Page 219
Monitoring of Fusion by Contents Intermixing......Page 220
Lateral Diffusion Coefficients......Page 221
Lipid Phase Transition......Page 223
Thermal Effects as Monitored by Differential Scanning Calorimetry......Page 224
Annexin Secondary Structure in Membrane-Bound State as Monitored by Circular Dichroism......Page 225
Monitoring Conformational Effects by Fluorescence......Page 226
Concluding Remarks......Page 228
Acknowledgments......Page 229
Glycosylphosphatidylinositol-Proteins and Interest in Membrane Reconstitutions......Page 230
Necessity to Control Membrane Orientation......Page 231
Description of Methods......Page 232
Preparation of GPI-Protein......Page 233
Determination of Liposome Stability......Page 235
Insertion of Lipoconjugates......Page 237
Characterization of Reconstituted Membranes......Page 239
Acknowledgments......Page 246
Introduction......Page 247
Equations for Competitive Binding of Particles to Several Types of Cells or Receptors in Absence of Endocytosis......Page 248
Binding and Cell Entry......Page 250
Kinetics of Binding and Endocytosis: Examples......Page 251
Endocytosis via Coated Pits......Page 254
Endocytosis via Caveolae......Page 257
Inhibitors of Endocytosis via Coated Pits......Page 258
Inhibitors of Caveolae......Page 259
Acknowledgments......Page 260
Introduction......Page 261
Lipoprotein-Containing Liposomes......Page 267
Incubation of Liposomes with Cytosol and Nucleotides......Page 269
Electron Microscopy......Page 270
Lipid Biochemistry: Measurement of Phosphoinositide Levels......Page 271
Concluding Remarks......Page 273
Introduction......Page 274
Terbium-Dipicolinic Acid Assay......Page 275
ANTS-DPX Assay......Page 279
NBD-Rhodamine Resonance Energy Transfer Assay......Page 281
Inner Monolayer Mixing Assay......Page 284
Leakage of Contents......Page 285
Carboxyfluorescein or Calcein Leakage......Page 286
ANTS-DPX Leakage......Page 287
Concluding Remarks......Page 288
Introduction......Page 289
General Comments......Page 292
Production of Snc2p-His6......Page 295
Cell Growth......Page 296
Affinity Purification......Page 297
Production of His8-Sso1p......Page 298
Production of GST-Sec9c......Page 299
Lipid Stocks......Page 300
Lipid Resuspension......Page 301
Dialysis......Page 302
Gradient Preparation......Page 303
Gradient Harvesting......Page 304
Electron Microscopy......Page 306
Equipment......Page 307
Data Analysis......Page 308
‘‘Rounds of Fusion’’ Calculations......Page 309
Conclusions......Page 313
Future Directions......Page 314
Acknowledgments......Page 315
Introduction......Page 316
Calibration of Buffer K......Page 319
Evaporation......Page 320
Extrusion......Page 321
Octadecyl Rhodamine......Page 322
NBD-Rhodamine-PE......Page 324
Specific Methods......Page 325
ANTS–DPX System I: For Two Liposomes......Page 326
Specific Methods......Page 327
ANTS–DPX System II: For Two Generic Vesicles......Page 328
Specific Methods......Page 329
Target Membranes and Vesicles for Neutrophil Fusion Studies......Page 331
Specific Methods......Page 332
Conclusions......Page 334
Introduction......Page 336
Modified Nonconventional Liposome Formulations for Cytosolic Delivery......Page 338
New Liposome Formulation Mimicking the Intracellular Bacterium Listeria monocytogenes......Page 339
Liposomes Containing LLO: Listeriosomes......Page 341
Purification of Recombinant Listeriolysin O......Page 342
Monitoring Cytosolic Delivery by Morphological Assay Using Fluorescent Dye......Page 343
Encapsulation of HPTS in Listeriosomes......Page 344
In Vitro Cytosolic Delivery of Macromolecule Antigen in Antigen-Presenting Cells......Page 347
51 Cr Release Assay for Antigen-Specific Cytotoxic T Lymphocyte Activity......Page 349
Antigen-Specific Interferon   Production as Measure of Cell-Mediated Immune Response Primed by in Vivo Cytosolic Delivery......Page 351
Encapsulation of Oligonucleotide and LLO into Liposomes......Page 352
Determination of Protein Expression by Flow Cytometric Analysis......Page 353
Determination of mRNA Levels by Northern Blot Analysis......Page 354
Concluding Remarks......Page 355
Acknowledgments......Page 356
Introduction......Page 357
Reagents......Page 358
Synthesis of pNP-PEG-DOPE......Page 359
Attachment of Tat Peptide to Liposomes......Page 360
Intracellular Localization and Trafficking of Tat-Liposomes......Page 362
Temperature Dependence of Liposome Uptake......Page 365
Concluding Remarks......Page 366
Endosomal Escape......Page 368
Isolation of Rat Peritoneal Macrophages......Page 370
Intracellular Trafficking of Macromolecules Encapsulated in Liposomes......Page 371
Effect of pH-Sensitive Liposomes on Intracellular Degradation of Encapsulated Albumin in Rat Peritoneal Macrophages......Page 372
Confocal Microscopic Analysis of Manipulation of Intracellular Trafficking with pH-Sensitive Liposomes......Page 374
Nuclear Transport of Macromolecules......Page 375
Conjugation of NLS Segment to Cargo Protein......Page 376
Intracellular Trafficking of Nuclear-Targeted Macromolecules Encapsulated into Liposomes......Page 377
Acknowledgments......Page 380
Introduction......Page 381
Preparation of Liposomes......Page 382
Determining Ability of Virus-Derived Peptide to Cause Fusion of Liposomes......Page 383
Dissipation of Diffusion Potential......Page 384
Calcein Release......Page 385
Determination of Bilayer Mixing by Using Fluorescence Probe Dilution Assay......Page 387
Determination of Hemifusion by Fluorescence Probe Dilution of Liposomes Pretreated with Dithionite......Page 391
Determination of Fusion of Liposomes by Electron Microscopy......Page 392
Concluding Remarks......Page 393
Introduction......Page 394
Binding Assays......Page 395
Lipid-Mixing Assays......Page 396
Production and Characterization of SINV or SFV......Page 397
Production of 35 Smethionine-Labeled Virus......Page 398
Preparation of Liposomes......Page 399
Protocol for Binding of 35 Smethionine-Labeled Virions to Liposomes......Page 400
Protocol for Fusion of Pyrene-Labeled SINV or SFV with Liposomes......Page 401
Protocol for Fusion of Unlabeled SINV or SFV with pyrPC-Labeled Liposomes......Page 402
Protocol for Fusion of 35Smethionine-Labeled SINV or SFV Particles with Trypsin-Containing Liposomes......Page 403
Identifying Target Membrane Lipids Involved in Alphavirus Fusion......Page 405
Lack of Involvement of Sphingolipid/Cholesterol Microdomains in Alphavirus Membrane Fusion......Page 407
Virus-Receptor Interaction and Its Influence on Membrane Fusion Activity......Page 410
Concluding Remarks......Page 411
Acknowledgments......Page 412
Introduction......Page 413
Lipid-Mixing Assays......Page 414
Content-Mixing Assays......Page 416
Kinetics of Fusion Studied with Liposomes......Page 417
Characterization of Fusion Pathway Using Lysophosphatidylcholine and Cis-Unsaturated Fatty Acids......Page 418
Trapping Fusion Intermediates......Page 425
Evolution and Stability of Captured Intermediates......Page 427
Acknowledgments......Page 428
Introduction......Page 429
Determination of Pore Size......Page 430
Determination of Number of Fusion Protein Ectodomains Involved in Pore Formation by Soluble Ectodomain of HA......Page 433
Transfer of Liposome-Entrapped Fluorescent Probes as a Result of Fusion versus Leakage......Page 434
Measuring Time Course of Pore Formation......Page 435
Transmembrane Movement of Fluorescent Phospholipids versus Entry of Dithionite through Pores......Page 437
Conclusions......Page 438
Acknowledgments......Page 439
Introduction......Page 440
Construction of Recombinant Baculovirus......Page 442
Amplification of Recombinant Baculovirus......Page 443
Purification of Envelope Protein by IMAC......Page 444
Reconstitution of Purified Envelope Proteins......Page 445
Binding Protocols and Detection......Page 446
Production and Purification of Viral Envelope Proteins......Page 447
Incorporation of E1 and E2 into Liposomes......Page 448
Surface Display of E1 and E2 Proteins on Liposomes......Page 449
Acknowledgment......Page 450
Introduction......Page 451
EnvA-PI......Page 456
EnvA-PI-Liposome Binding Procedure I......Page 457
EnvA-PI-Liposome Binding Procedure II......Page 458
Conclusions......Page 460